Week 14:
worked on enzyme assays and inhibition assays
Week 13:
This week I worked on FPLC and we concentrated the protein. We also worked on an enzyme assay on J.C. enzyme. I redid the homology model and I will start the virtual screening.
dg26682_divyaFPLC.JPG
FTHAP FPLC- Tubes 30-38 are contaminated
js64927_proteinafterFPLC.jpg
Protein concentrated after FPLC to 1mL
111712_JongguShin_FPLCbeforeConc.jpg
nanodrop results before concentrating
Lane 1: n/a Lane 2: ladder Lane 3: sample 1 Lane 4: n/a Lane 5: sample 3 Lane 6: sample 4 Lane 7: n/a Lane 8: elution 1 Lane 9: elution 2 Lane 10: n/a
112612 - for Week 13 - do you have some virtual results. Dr B
Week 12:
this week I worked on characterization and purification protocol.
Ok - Dr. B 11/19/12
Week 11: I Continued working on Homology Model and starting the virtual Screening. Finished Protein Expression of FTHAP worked on Material and Methods for cloning
Week 10: This week I worked on making PNIC BSA-4 for cloning. The concentration however was not large enough. I then started to work on the surrogate protein expression FTHAP on thursday and friday. Nanodrop for PNIC BSA4 for cloning
Week 9: This week I worked on running the gel for my PCR and also redoing PCR since the band is low. I also worked on the research paper
Lane 1- skip
Lane 2- 1 Kb ladder
Lane 3- PCR 1
Lane 4- Secondary PCR with annealing 55 degrees
Lane 5- Secondary PCR with annealing 57 degrees Week 8: 102112 - Sajan, show some results here. - Dr. B
This week I worked on virtual screening refresher. I also worked on PCR secondary. Week 7:
101612 - Sajan - so, I dont think your PCR^2 is actually good. Your bands should be the size of your gene. If you are using a 100 bp ladder - then these seem way too low. --Dr. B
This week I worked on making the primer dilution which tom ordered. I made a oligo mix which was used to do primary and secondary PCR for my target. I ran a gel for the primary and secondary PCR. I also did PCR Squared for my target. All of the gels looked good and I will proceed to PCR cleanup. I also worked on starting my virtual screening of my target by doing a homology model and then proceeding to screen from that,
Lane 1: skip
Lane 2: 100 bp Ladder
Lane 3: PCR 1
Lane 4: PCR 2
Lane 1: skip
Lane 2: 100 bp ladder
Lane 3: PCR squared 1
Lane 4: PCR Squared 2
Lane 5 :PCR squared 3
Lane 6: PCR squared 4
Week 6: 100912 - Sajan, ok good. Show your gels for primary and secondary PCR. - DR. B This week I worked on running the gel from the primary and secondary PCR. The gels unfortunately failed. I also worked on doing my primer design. I started the PCR primer overlap. I worked on the primer dilution and PCR one and secondary PCR for the Primer Overlap.
Figure 1: agarose gel of pGBR22 with M13 primer.
Lane 2:100bp DNA ladder
Lane 7:1:1000 dilution
Lane 8:1:1000 dilution
Lane 9:1:100 dilution
Lane 10: no DNA control
Week 5:
093012 - sajan, missing Tail Primer Design. Good analysis on your gel. Should include results of your PCR's. Also, use the standard format for labeling your gel with each lane on a separate row. - Dr. B This week I worked on running the gel for restriction enzyme digest from the week 3 which I had not done. I also did PCR and PCR two this week. I have also finished doing Pymol Refresher this week. This is the gel run from the re digest. The Ladder is located on Lane two and the uncut plasmid is in lane three. Lane four through six contains the plasmid cut with ECO
RI, PVU II and both ECO RI and PVU II. The plasmid however could not be seen. This could be due to the length between the preparation and running the gel. The DNA
could have degraded while in the -20 freezer or there could be loss of the plasmid through not properly capping the tubes.
Week 4:
sajan - looks good. Dr. B This week I worked on Midi prep. I also did a nanodrop to find the concentration of sample to be 53.8 ng/uL.
the sample was also sent to DNA sequencing to determine the sequence so that we can proceed to cloning. Here is the forward sequencing
It was determine that it was the pNIC vector through a nucleotide blast.
Week 3:
Sajan - can you include an image of your RE digest with analysis? -- DR. B 091812
This week I worked on the RE digest along with doing transformation that will be later used for the midi prep next week.
Week 2:
This week I did primer dilution and the DNA sequencing. I did a forward primer dilution which was then used with a reverse primer for the submission to the DNA sequencing facility for pNIC-Bsa4 vector. On Friday I did a DNA sequence analysis protocol where we used the purple protein and did a protein blast. We also looked at restriction enzyme cuts on the computer.
Week 1:
This week I worked on finding a target for this semester. Here is my info from the target page.
*Background/Disease Information: C. perfringens is a bacteria that leads to necrosis, bacteremia and gas gangrene. It is the most common bacterial reagent leading to gas gangrene. It is the third most common cause of food poisoning in the United Kingdom and the United States.
Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens.
-- PDB # or closest PDB entry if using homology model: 2WXT
Inhibitors
Inhibitors
Commentary
Organism
Structure
EDTA
enzyme activity against 1-myristoyl-lysophosphatidylcholine, glycerophosphorylcholine, and p-nitrophenyl phosphorylcholine are readily inhibited by EDTA. Indicates that a divalent cation is essential for catalytic activity
enzyme activity against 1-myristoyl-lysophosphatidylcholine, glycerophosphorylcholine, and p-nitrophenyl phosphorylcholine are readily inhibited by EGTA. Indicates that a divalent cation is essential for catalytic activity
the catalytic activity of NPP6 toward p-nitrophenyl phosphorylcholine is partially inhibited by the divalent cations. Attempts are unsuccessful to recover the activity of EDTA treated NPP6 by adding various divalent cations
Homo sapiens
-
Expression Information (has it been expressed in bacterial cells): homo sapiens, E. Coli and mus musculus. Yes it was expressed in E. Coli
Purification Method:
mono Q ion exchange chromatography column and eluted with a linear gradient of NaCl (0-2 M) using the deltaKTA system in homo sapiens and mus musculus
worked on enzyme assays and inhibition assays
Week 13:
This week I worked on FPLC and we concentrated the protein. We also worked on an enzyme assay on J.C. enzyme. I redid the homology model and I will start the virtual screening.
FTHAP FPLC- Tubes 30-38 are contaminated
Protein concentrated after FPLC to 1mL
nanodrop results before concentrating
Lane 1: n/a
Lane 2: ladder
Lane 3: sample 1
Lane 4: n/a
Lane 5: sample 3
Lane 6: sample 4
Lane 7: n/a
Lane 8: elution 1
Lane 9: elution 2
Lane 10: n/a
112612 - for Week 13 - do you have some virtual results. Dr B
Week 12:
this week I worked on characterization and purification protocol.
Ok - Dr. B 11/19/12Week 11:
I Continued working on Homology Model and starting the virtual Screening.
Finished Protein Expression of FTHAP
worked on Material and Methods for cloning
Week 10:
This week I worked on making PNIC BSA-4 for cloning. The concentration however was not large enough. I then started to work on the surrogate protein expression FTHAP on thursday and friday.
Nanodrop for PNIC BSA4 for cloning
Week 9:
This week I worked on running the gel for my PCR and also redoing PCR since the band is low. I also worked on the research paper
Lane 1- skip
Lane 2- 1 Kb ladder
Lane 3- PCR 1
Lane 4- Secondary PCR with annealing 55 degrees
Lane 5- Secondary PCR with annealing 57 degrees
Week 8:
102112 - Sajan, show some results here. - Dr. B
This week I worked on virtual screening refresher. I also worked on PCR secondary.
Week 7:
101612 - Sajan - so, I dont think your PCR^2 is actually good. Your bands should be the size of your gene. If you are using a 100 bp ladder - then these seem way too low. --Dr. B
This week I worked on making the primer dilution which tom ordered. I made a oligo mix which was used to do primary and secondary PCR for my target. I ran a gel for the primary and secondary PCR. I also did PCR Squared for my target. All of the gels looked good and I will proceed to PCR cleanup. I also worked on starting my virtual screening of my target by doing a homology model and then proceeding to screen from that,
Lane 1: skip
Lane 2: 100 bp LadderLane 3: PCR 1
Lane 4: PCR 2
Lane 1: skip
Lane 2: 100 bp ladder
Lane 3: PCR squared 1
Lane 4: PCR Squared 2
Lane 5 :PCR squared 3
Lane 6: PCR squared 4
Week 6:100912 - Sajan, ok good. Show your gels for primary and secondary PCR. - DR. B
This week I worked on running the gel from the primary and secondary PCR. The gels unfortunately failed. I also worked on doing my primer design. I started the PCR primer overlap. I worked on the primer dilution and PCR one and secondary PCR for the Primer Overlap.
Figure 1: agarose gel of pGBR22 with M13 primer.
Lane 2:100bp DNA ladder
Lane 7:1:1000 dilution
Lane 8:1:1000 dilution
Lane 9:1:100 dilution
Lane 10: no DNA control
Week 5:
093012 - sajan, missing Tail Primer Design. Good analysis on your gel. Should include results of your PCR's. Also, use the standard format for labeling your gel with each lane on a separate row. - Dr. BThis week I worked on running the gel for restriction enzyme digest from the week 3 which I had not done. I also did PCR and PCR two this week. I have also finished doing Pymol Refresher this week.
This is the gel run from the re digest. The Ladder is located on Lane two and the uncut plasmid is in lane three. Lane four through six contains the plasmid cut with ECO
RI, PVU II and both ECO RI and PVU II. The plasmid however could not be seen. This could be due to the length between the preparation and running the gel. The DNA
could have degraded while in the -20 freezer or there could be loss of the plasmid through not properly capping the tubes.
Week 4:
sajan - looks good. Dr. BThis week I worked on Midi prep. I also did a nanodrop to find the concentration of sample to be 53.8 ng/uL.
the sample was also sent to DNA sequencing to determine the sequence so that we can proceed to cloning. Here is the forward sequencing
It was determine that it was the pNIC vector through a nucleotide blast.
Week 3:
Sajan - can you include an image of your RE digest with analysis? -- DR. B 091812This week I worked on the RE digest along with doing transformation that will be later used for the midi prep next week.
Week 2:
This week I did primer dilution and the DNA sequencing. I did a forward primer dilution which was then used with a reverse primer for the submission to the DNA sequencing facility for pNIC-Bsa4 vector. On Friday I did a DNA sequence analysis protocol where we used the purple protein and did a protein blast. We also looked at restriction enzyme cuts on the computer.Week 1:
This week I worked on finding a target for this semester. Here is my info from the target page.
*Target (protein/gene name): phospholipase C
*NCBI Gene # or RefSeq#: NCTC8237
GenBank: CAA31943.1
http://www.ncbi.nlm.nih.gov/protein/CAA31943.1
>gi|194293816|gb|EU839779.1| Clostridium perfringens strain S01 phospholipase C (plc) gene, complete cds
*Organism: Clostridium Perfrigens
*Background/Disease Information: C. perfringens is a bacteria that leads to necrosis, bacteremia and gas gangrene. It is the most common bacterial reagent leading to gas gangrene. It is the third most common cause of food poisoning in the United Kingdom and the United States.
Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens.
http://iai.asm.org/content/57/2/367.short
Complex of enzymes:
*EC#: 3.1.4.3
Link to BRENDA EC# page: http://brenda-enzymes.org/literature/lit.php4?e=3.1.4.3&r=665632
--Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/phospholipaseccper.Par.0001.File.tmp/phospholipaseccper.pdf
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2WXT
Inhibitors
Expression Information (has it been expressed in bacterial cells): homo sapiens, E. Coli and mus musculus. Yes it was expressed in E. Coli
Purification Method:
mono Q ion exchange chromatography column and eluted with a linear gradient of NaCl (0-2 M) using the deltaKTA system in homo sapiens and mus musculus
*Amino Acid Sequence: *
1 MSRLVVVSNRIAPPDEHAASAGGLAVGILGALKAAGGLWFGWSGETGNEDQPLKKVKKGN
61 ITWASFNLSEQDLDEYYNQFSNAVLWPAFHYRLDLVQFQRPAWDGYLRVNALLADKLLPL
121 LQDDDIIWIHDYHLLPFAHELRKRGVNNRIGFFLHIPFPTPEIFNALPTYDTLLEQLCDY
181 DLLGFQTENDRLAFLDCLSNLTRVTTRSAKSHTAWGKAFRTEVYPIGIEPKEIAKQAAGP
241 LPPKLAQLKAELKNVQNIFSVERLDYSKGLPERFLAYEALLEKYPQHHGKIRYTQIAPTS
301 RGDVQAYQDIRHQLENEAGRINGKYGQLGWTPLYYLNQHFDRKLLMKIFRYSDVGLVTPL
361 RDGMNLVAKEYVAAQDPANPGVLVLSQFAGAANELTSALIVNPYDRDEVAAALDRALTMS
421 LAERISRHAEMLDVIVKNDINHWQECFISDLKQIVPRSAESQQRDKVATFPKLA
Primer design
1 ATGTCTCGTCTCGTTGTTGTTTCTAATCGTATCGCGCCTCCAGACGAACACGCGGCGTC 59
2 GCCGCCTTGAGGGCACCGAGGATACCCACCGCGAGGCCACCAGCAGACGCCGCGTGTTCG 60
3 TGCCCTCAAGGCGGCAGGCGGTCTGTGGTTCGGTTGGTCCGGTGAGACCGGTAACGAGGA 60
4 CCCAGGTGATGTTACCTTTCTTAACTTTTTTGAGTGGCTGATCCTCGTTACCGGTCTCAC 60
5 AGAAAGGTAACATCACCTGGGCGTCTTTCAACCTGTCTGAACAAGACCTGGACGAATACT 60
6 GGAACGCAGGCCAGAGAACGGCGTTAGAGAACTGGTTGTAGTATTCGTCCAGGTCTTGTT 60
7 TCTCTGGCCTGCGTTCCATTACCGTCTCGACCTGGTGCAATTTCAGCGTCCAGCGTGGGA 60
8 GGGAGGAGTTTATCCGCCAGCAGCGCGTTAACACGCAGATAGCCGTCCCACGCTGGACGC 60
9 TGGCGGATAAACTCCTCCCGCTCCTCCAGGACGACGATATCATCTGGATTCACGACTACC 60
10 CGCCACGCTTACGCAGCTCGTGCGCGAACGGCAGGAGGTGGTAGTCGTGAATCCAGATGA 60
11 CTGCGTAAGCGTGGCGTAAATAACCGTATCGGTTTCTTCCTGCACATCCCGTTCCCAACC 60
12 CAGCAGGGTGTCGTAGGTCGGGAGTGCGTTAAAGATTTCCGGGGTTGGGAACGGGATGTG 60
13 ACCTACGACACCCTGCTGGAACAGCTCTGTGACTACGACCTGCTCGGCTTCCAGACCGAG 60
14 GGGTCAGATTAGAGAGGCAGTCCAGAAACGCCAGACGGTCATTCTCGGTCTGGAAGCCGA 60
15 CTGCCTCTCTAATCTGACCCGTGTTACCACTCGTAGCGCGAAATCTCATACTGCGTGGGG 60
16 TTCGATACCGATAGGGTAAACTTCGGTGCGGAACGCTTTACCCCACGCAGTATGAGATTT 60
17 AAGTTTACCCTATCGGTATCGAACCGAAAGAAATCGCGAAACAAGCAGCCGGTCCGCTGC 60
18 TTCTGAACGTTTTTCAGTTCCGCTTTGAGCTGGGCCAGCTTTGGTGGCAGCGGACCGGCT 60
19 CGGAACTGAAAAACGTTCAGAACATCTTCTCTGTTGAACGCCTGGACTACTCTAAAGGTC 60
20 TTTCCAGGAGCGCTTCGTACGCCAGGAAACGCTCCGGCAGACCTTTAGAGTAGTCCAGGC 60
21 ACGAAGCGCTCCTGGAAAAATACCCGCAACATCATGGTAAAATTCGTTACACCCAGATTG 60
22 CTTGGTACGCCTGCACGTCGCCACGAGAGGTCGGCGCAATCTGGGTGTAACGAATTTTAC 60
23 CGTGCAGGCGTACCAAGACATCCGTCACCAACTGGAGAACGAAGCAGGCCGCATCAACGG 60
24 GGTTCAGGTAGTACAGCGGAGTCCAACCCAGCTGACCGTACTTACCGTTGATGCGGCCTG 60
25 CCGCTGTACTACCTGAACCAACACTTCGACCGTAAACTGCTGATGAAAATCTTCCGTTAC 60
26 TACCGTCGCGCAGTGGGGTCACCAGACCAACGTCAGAGTAACGGAAGATTTTCATCAGCA 60